The present invention is concerned with a new family of synaptically activated proteins, and in particular, a protein that specifically binds to and alters the function of metabotropic glutamate receptors.
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Spatial localization and clustering of membrane proteins is critical to neuronal development and synaptic plasticity. Proteins that interact with plasma membrane proteins are thought to affect the spatial distribution of such membrane proteins. These interactions are may be important in regulating the function(s) of membrane proteins, such as neurotransmitter receptors, which control synaptic activity in the central nervous system.
The present invention concerns the discovery of a new family of proteins that are enriched in the mammalian central nervous system and that interact with proteins involved in synaptic function. These proteins are involved in synaptic function, as evidenced by their induction by neuronal activation, such as seizures, visual stimulation, acute cocaine, trauma, and the like. These proteins are collectively termed xe2x80x9csynaptic activation proteins.xe2x80x9d
A novel dendritic protein, termed xe2x80x9cHomerxe2x80x9d, exemplifies the present invention. This protein contains a single, PDZ-like binding domain and binds specifically to the C-terminus of metabotropic glutamate receptors. Metabotropic glutamate receptors release intracellular calcium by activating phospholipase C, which catalyzes the hydrolysis of membrane phosphoinositides. However, other than containing a PDZ-like domain, the Homer protein does not otherwise resemble known PDZ proteins and has less than 10% sequence identity with the closest PDZ protein. Additionally, the Homer protein is regulated as an immediate early gene. This dynamic transcriptional control suggests that Homer mediates a novel cellular mechanism to regulate metabotropic glutamate signaling.
The features outlined for the Homer protein characterize a novel family of proteins, synaptic activation proteins, which form the basis for the present invention. Because these proteins are involved in synaptic function, they have particular utilities, for example, in screening assays for drugs that affect synaptic function and are therefore centrally active, as further described below.
The present invention is concerned with a novel family of proteins that are present in the mammalian central nervous system. These proteins are particularly characterized by (i) their enhanced expression in mammalian central nervous tissue in response to synaptic activation, and (ii) a novel PDZ-like binding domain.
The new protein family is exemplified by a rat protein, termed xe2x80x9cHomerxe2x80x9d (SEQ ID NO: 2). Other members of the family have a sequence that is substantially identical (e.g., at least 70% or greater sequence identity, and preferably 80% or greater sequence identity) to that of the rat protein, or to the human and mouse members of the family, setments of which are shown herein as SEQ ID NO: 3 and SEQ ID NO: 4. The full length versions of these latter peptides, insofar as they are revealed by the discovery of the full-length sequences described herein, also form part of the present invention. The present invention also include species homologs and/or compositions based on internally consistent variations between SEQ ID NO: 2 and such full length species homologs, as described herein.
More specifically, proteins having sequences that comprise internally consistent variants among the disclosed sequences, including conservative amino acid substitutions thereof, also form a part of the invention. Family members may be identified by low stringency hybridization, degenerate PCR, or other methods that detect nucleotide or amino acid sequences that are at least about 70%, and in a preferred embodiment at least 80%, identical to SEQ ID NO: 1 or SEQ ID NO: 2, respectively.
Proteins of the invention are particularly useful for use in screening assays for centrally active drugs. The proteins are also useful components of diagnostic assays for measuring induction of synaptic activation, as may occur in response to multiple stimuli that result in synaptic activation. Similarly, peptide fragments from such proteins, particularly peptide fragments derived from the binding site between such proteins and their synaptic effector binding partners, have utility as inhibitors long term consequences of abnormal synaptic activation.
In a related embodiment, the invention includes polypeptides as described above, but which further exhibit an ability to selectively bind to a synaptic membrane protein having a C-terminal peptide region selected from the group consisting of SSSL (SEQ ID NO: 11) and SSTL (SEQ ID NO: 10.
In a related aspect, the invention also includes nucleotide sequences that encode members of the novel protein family described herein. Accordingly, nucleotide sequences having substantial identity to the disclosed Homer protein coding sequences (such as SEQ ID NO: 1), as well as the disclosed sequences themselves, are also included within the invention.
Also forming a part of the invention are vectors containing the polynucleotide sequences described above. Such vectors are useful, for example, in the production of the claimed proteins by recombinant techniques.
In a related aspect, the invention also includes a method of selecting a compound that interferes with binding of a synaptic activation protein to a cellular binding protein in the mammalian central nervous system. The method includes adding a test compound to a reaction mixture containing (i) an isolated synaptic activation protein having substantial identity to one or more of the polypeptides having the substantial sequence identity to the polypeptide having a sequence presented as SEQ ID NO: 2, (ii) an isolated binding protein to which the synaptic activation protein binds, and (iii) means for detecting binding between the synaptic activation protein and said binding protein. Binding of the binding protein to the synaptic activation protein is measured in the presence of a test compound and compared to binding measured in the absence of the test compound. A test compound is selected for use as a centrally active drug if such comparison reveals a substantial difference in binding under these conditions.
In a particular embodiment, the binding protein in the assay method is a mebotropic glutamate receptor polypeptide which includes a sequnce selected from the group the group consisting of SSSL (SEQ ID NO: 11) and SSTL (SEQ ID NO: 10). In another particular embodiment, the binding protein is an mGluR linked to phosphoinositidase C. In yet another embodiment. mGluR is expressed in cells, and binding between the receptor and the binding protein is measured by measuring phosphoinositidase C activity in cells.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.